The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-regulated chloride channel that determines the rate of electrolyte and fluid transport in the apical membrane of epithelial cells. Abnormal CFTR function is associated with the pathogenesis of cystic fibrosis and secretory diarrhea. Our long-term objective is to understand the molecular mechanisms underlying the regulation of CFTR at the atomic level and develop novel strategies for modulating the activity of this channel and treating the CFTR-associated diseases. The CFTR topology consists of two membrane-spanning domains and five cytoplasmic domains: an N-terminal domain (NTD), two nucleotide-binding domains, a regulatory domain (R) and a C-terminal domain (CTD). The CFTR activity is modulated through phosphorylation of the R domain, ATP hydrolysis by the NBDs, and interactions of its NTD and CTD domains with syntaxin 1A and NHERF proteins, respectively. However the regulatory mechanisms remain unknown primarily because the three-dimensional structure of the CFTR domains and the structural basis of their interaction with intracellular regulatory proteins remain elusive. This proposal addresses these questions and focuses on the structural analysis of cytoplasmic CFTR domains and their complexes with regulatory proteins, using molecular biology techniques and X-ray crystallography. The specific aims are: 1. To dissect the structural basis of CFTR channel gating mediated through the interaction of the CFTR CTD with the NHERF PDZ1 and PDZ2 domains. 2. To elucidate the molecular mechanisms underlying the regulation of CFTR channel activity through the interaction of the CFTR NTD with syntaxin 1A. 3. To determine the three-dimensional atomic structures of the CFTR NBD1 and NBD2 domains. These studies will provide the first high-resolution three-dimensional structures of four cytoplasmic CFTR domains and the structural basis of CFTR regulation by proteins syntaxin 1A and NHERF. This information is an essential step towards elucidating the basic molecular mechanisms that control the CFTR channel gating. Importantly, the atomic coordinates of these complexes could be used for structure-based rational design of drugs that would modify selectively the CFTR activity with clinical applications in the treatment of cystic fibrosis and secretory diarrhea.